The stable propagation of our genomes requires equal partitioning of DMA into daughter cells by a microtubule-based bipolar spindle. Errors in cell division are associated with developmental defects and disease. Our long-term goal is to determine the molecular basis for bipolar spindle assembly during mammalian mitosis. We propose an interdisciplinary approach to examine how microtubule transport and polymerization contribute to bipolar spindle assembly, focusing on kinesin-5 and Polo-like kinases (Plks), evolutionarily conserved proteins whose loss-of-function leads to monopolar spindles. Mitotic spindle assembly requires kinesin-5 dependent crosslinking and sliding of microtubules. Single molecule studies have characterized kinesin-5 motility on individual microtubules. However, the motile properties of kinesin-5 while crosslinking two microtubules remain unknown. In addition, the contribution of kinesin-5's homo- tetrameric structure, which is essential for microtubule crosslinking, remains poorly understood. Recently, we and others have characterized chemical inhibitors for Plks. When combined with high-resolution microscopy, these inhibitors can be powerful tools to examine cell division dynamics. Similar to Plk1 knockdown, Plk inhibitor-treated cells form monopolar spindles that lack proper centrosome function. Failure in centrosome separation, as observed upon kinesin-5 inhibition, is not likely to be the cause of monopolar spindles in cells lacking Plk function as centrosomes can be separated by distances comparable to that in normal metaphase cells. We have also found that spindle collapse upon Plk inhibition is associated with kinetochore fiber shortening and the appearance of persistent non-kinetochore fibers. These findings suggest that Plk- dependent regulation of microtubule polymerization is needed for bipolar spindle formation. In this project we will: (1) Examine the motility of single Eg5, vertebrate kinesin-5, molecules between two microtubules. (2) Determine how Eg5's structural organization influences its motile properties and function. (3) Examine the role of Polo-like kinases in regulating spindle microtubule dynamics. (4) Characterize Polo-like kinase substrates that regulate microtubule dynamics in dividing cells. Chemical inhibitors of Eg5 and Polo-like kinases are currently in clinical trials as anti-cancer agents. Our studies will provide insight into the cell division functions of these proteins and also impact the development of drugs that target them.